TWI570240B - A conditional replicating cytomegalovirus as a vaccine for cmv - Google Patents

Description

Conditional replication CMV as a cellular giant virus vaccine

The present invention relates to a method for inducing an immune response to CMV using genetically modified conditionally replicating defective cell giant virus (CMV). The invention also relates to CMVs that have been recombined to allow external control of viral replication. Compositions comprising the replication defective CMV are also contemplated by the present invention.

The cell giant virus (CMV) (also known as human herpesvirus 5, HHV-5) is a herpes virus classified as a member of the beta subfamily of herpesviridae. According to the Centers for Disease Control and Prevention, CMV infection is fairly widespread in the human population, with an estimated 40-80% of the adult population in the United States being infected. The virus is primarily transmitted through body fluids and is often delivered from a pregnant mother to a fetus or newborn. In most individuals, CMV infection is latent, but viral activation can lead to high fever, chills, fatigue, headache, nausea, and splenomegaly.

Although most human CMV infections are asymptomatic, CMV infection in immunocompromised individuals (such as HIV-positive patients, allograft patients, and cancer patients) or those whose immune system is not fully developed (eg, neonates) can be particularly problematic (Mocarski et al. , Cytomegalovirus, in Field Virology, 2701-2772, edited by Knipes and Howley, 2007). CMV infection in these individuals can cause serious conditions including pneumonia, hepatitis, encephalitis, colitis, uveitis, retinitis, blindness and neuropathy, as well as other harmful conditions. In addition, CMV infection during pregnancy is the main cause of birth defects (Adler, 2008 J. Clin Virol, 41: 231; Arvin et al., 2004 Clin Infect Dis, 39: 233; Revello et al., 2008 J Med Virol, 80: 1415). CMV infects a variety of living cells, including mononuclear cells, macrophages, dendritic cells, neutrophils, endothelial cells, epithelial cells, fibroblasts, neurons, smooth muscle cells, hepatocytes, and stromal cells (Plachter et al. 1996, Adv. Virus Res. 46: 195). Although clinical CMV isolates replicated in a variety of cell types, laboratory strains AD 169 (Elek and Stern, 1974, Lancet 1:1) and Towne (Plotkin et al., 1975, Infect. Immun. 12: 521) are almost exclusively Replication in fibroblasts (Hahn et al., 2004, J. Virol. 78: 10023). It is stipulated that the continuation of continuous passage and final adaptation of the virus in fibroblasts is limited to attenuation markers (Gerna et al., 2005, J. Gen. Virol. 86: 275; Gerna et al., 2002, J. Gen. Virol. 83: 1993; Gerna et al, 2003, J. Gen Virol. 84: 1431; Dargan et al, 2010, J. Gen Virol. 91:1535). Mutations in the tropism of epithelial cells, endothelial cells, white blood cells and dendritic cells in human CMV laboratory strains have been mapped to three open reading frames (ORF): UL128, UL130 and UL131 (Hahn et al., 2004, J). .Virol. 78:10023; Wang and Shenk, 2005 J. Virol. 79: 10330; Wang and Shenk, 2005 Proc Natl Acad Sci USA. 102: 18153). Biochemical and remodeling studies have shown that UL128, UL130 and UL131 are assembled onto gH/gL scaffolds to form pentameric gH complexes (Wang and Shenk, 2005 Proc Natl Acad Sci USA. 102: 1815; Ryckman et al., 2008 J. Virol) .82:60). Recovery of this complex in virions restored viral epithelial tropism in laboratory strains (Wang and Shenk, 2005 J. Virol. 79: 10330).

Endothelial and epithelial tropism has been previously assessed as a defect in vaccines such as Towne (Gerna et al, 2002, J. Gen Virol. 83: 1993; Gerna et al, 2003, J. Gen Virol. 84: 1431). In the serum of human subjects infected with natural CMV, the activity of neutralizing antibodies against viral epithelial entry was 15 times that of fibroblasts (Cui et al, 2008 Vaccine 26: 5760). Humans with primary infections rapidly develop neutralizing antibodies against the viral endothelium and epithelium, but only slowly produce neutralizing antibodies against viral fibroblasts (Gerna et al., 2008 J. Gen. Virol. 89: 853). In addition, there is no neutralizing activity against viral epithelium and endothelium in the immune sera of human subjects receiving the Towne vaccine (Cui et al., 2008 Vaccine 26: 5760). Recently, a panel of human monoclonal antibodies from four donors with HCMV infection was described, and from this group, the homologous recognition of the pentameric gH complex antigen was high (Macagno et al., 2010 J. Virol. 84 :1005).

The present invention relates to the use of conditional replication-deficient CMV (rdCMV) and rdCMV in compositions and methods for treating a CMV infection in a patient or a condition associated with such an infection and/or reducing the likelihood thereof. The rdCMVs described herein comprise nucleic acids encoding one or more fusion proteins comprising essential proteins fused to a destabilizing protein. The fusion protein degrades in the absence of a stabilizer. Thus, rdCMV can be grown in tissue culture under conditions that permit replication (i.e., in the presence of a stabilizer), but reduces and preferably prevents replication when administered to a patient (in the absence of a stabilizer).

One embodiment of the invention is a conditional replication defective CMV. rdCMV A nucleic acid encoding one or more fusion proteins comprising an essential protein fused to a destabilizing protein is included. A nucleic acid encoding a wild-type essential protein is no longer present in rdCMV and thus the fusion protein is required for viral replication. In a preferred embodiment, the essential protein is selected from the group consisting of IE1/2, UL51, UL52, UL79, and UL84 and the destabilizing protein line FKBP or a derivative thereof.

Another embodiment of the invention is a composition comprising isolated rdCMV and a pharmaceutically acceptable carrier. The composition may further comprise an adjuvant including, but not limited to, an ISCOMATRIX® adjuvant and an aluminum phosphate adjuvant.

Another embodiment of the invention is the use of a rdCMV composition to induce an immune response against CMV in a patient. The patient can be treated prophylactically or therapeutically by administering the rdCMV of the invention. Prophylactic treatment provides sufficient protective immunity to reduce the likelihood or severity of CMV infection, including primary infections, recurrent infections (ie, those from latent CMV reactivation), and reinfection (ie, They originate from those infected with CMV different from those previously experienced by the patient). In a specific embodiment, women of childbearing age, particularly early adolescent women, are vaccinated to reduce the likelihood of CMV infection (primary, relapsing or re-) during pregnancy and thus reduce the likelihood of infecting CMV to the fetus. Therapeutic treatment can be performed to reduce the duration/severity of current CMV infections.

Another embodiment of the invention is a method of making a rdCMV of the invention comprising propagating rdCMV on epithelial cells (e.g., ARPE-19 cells, ATCC Accession No. CRL-2302) in the presence of Shield-1. In some embodiments, rdCMV is propagated in epithelial cells on a microcarrier or other high density cell culture system.

The present invention relates to the use of conditional replication-deficient CMV (rdCMV) and rdCMV in compositions and methods for treating a CMV infection in a patient or a condition associated with such an infection and/or reducing the likelihood thereof. The rdCMV described herein encodes one or more fusion proteins comprising an essential protein fused to a destabilizing protein, rather than a wild type essential protein. In the absence of a stabilizer, the fusion protein is degraded by the host cell machinery. In the presence of a stabilizer, the fusion protein is stable and does not degrade.

Suitable fusion proteins for use in the present invention retain sufficient essential protein activity in the presence of a stabilizer to promote viral replication in a host cell and reduce CMV replication in the absence of a stabilizer (preferably greater than 50%, 75%, 90%, 95% or 99% reduction). Preferably, the essential protein used in the fusion protein encodes a non-structural protein and is therefore not encapsulated into the rdCMV virion. Suitable essential proteins identified herein include CMV proteins encoded by the essential genes IE1/2, UL51, UL52, UL79, and UL84.

Examples of destabilizing proteins and stabilizers are set forth in U.S. Patent Publication No. 2009/0215169, which discloses compositions, systems and methods for the use of small molecule regulatory proteins. Briefly, the protein is fused to a protein FKBP or a derivative thereof that affects stability. The exogenously added cell-permeable small molecule Shield-1 (Shld-1) interacts with FKBP or its derivatives and stabilizes the fusion protein. In the absence of Shield-1, FKBP or its derivatives direct the fusion protein to be degraded by the host cell machinery.

In an embodiment of the invention, the essential CMV protein is fused to FKBP or a derivative thereof. The fusion protein is stable in the presence of Shield-1. however, In the absence of Shield-1, FKBP or its derivatives direct the fusion protein to be degraded by the host cell machinery.

In the absence of the fusion protein, it is reduced (more preferably greater than 50%, 75%, 90%, 95% or 99% compared to CMV without the destabilizing essential protein) or prevents rdCMV replication.

The recombinant virus to be used in the method of the invention also displays an immunogenic pentameric gH complex on its virions.

Embodiments also include recombinant CMVs or compositions thereof, or vaccines comprising or consisting of the CMVs described herein, for use in (i) for (a) therapy (eg, human); b) drugs; (c) inhibiting CMV replication; (d) treating or preventing CMV infection; or (e) treating or preventing CMV-related diseases or delaying their onset or progression, (ii) acting as an agent for the above, or (iii) for the preparation of a medicament for use in the above. In such uses, the recombinant CMV, its compositions, and/or vaccines comprising or consisting of such CMVs or compositions may optionally be combined with one or more antiviral agents (eg, antiviral compounds or antiviral immunoglobulins; Combination vaccines, as described below) are used in combination.

As used herein, "inducing an immune response" refers to the ability of a conditional replication-deficient CMV to produce an immune response in a patient, preferably a mammal, or a better human, wherein the response includes, but is not limited to, the production of specific binding. Preferably, the CMV and/or components (eg, antibodies) that activate T cells are neutralized. "Protective immune response" reduces the likelihood of a patient being infected with CMV (including primary, recurrent, and/or reinfection) and/or improves at least one condition associated with CMV infection and/or reduces the severity of CMV infection. / Duration of the immune response.

As used herein, "immunologically effective amount" refers to an amount of an immunological response that an immunogen can induce against CMV when administered to a patient, which protects the patient from CMV infection (including primary, recurrent, and/or reinfection) And/or improving at least one condition associated with CMV infection and/or reducing the severity/duration of CMV infection in the patient. This amount should be sufficient to significantly reduce the likelihood or severity of CMV infection. Animal models known in the art can be used to assess the protective effects of the administered immunogen. For example, immune sera or immune T cells of an individual who is administered an immunogen can be analyzed for antibody or cytotoxic T cell neutralizing ability or interleukin production ability of immune T cells. Analysis commonly used for such assessments includes, but is not limited to, virus neutralization assays, antiviral antigen ELISA, interferon gamma interleukin ELISA, interferon gamma ELISPOT, intracellular multicellular staining (ICS), and ⁵¹ chrome Release cytotoxicity assay. An animal stimulation model can also be used to determine the immunologically effective amount of the immunogen.

As used herein, "conditional replication-defective virus" refers to viral particles that can be replicated in certain environments, but not in other environments. In a preferred embodiment, the virus is rendered a conditional replication-deficient virus by destabilizing one or more proteins necessary for viral replication. Nucleic acids encoding wild-type, non-destable essential proteins are no longer present in conditional replication-deficient viruses. Under conditions in which one or more essential proteins are destabilized, the reduction in viral replication is preferably greater than 50%, 75%, 90%, 95%, 99%, or 100% compared to a virus that does not have a destabilizing essential protein. However, under conditions which stabilize the destabilizing essential protein, viral replication may be preferably at least 75%, 80%, 90%, 95%, 99% or 100% of the amount of replication of the CMV which does not contain the destabilizing essential protein. occur. In a more preferred embodiment, by destabilizing the protein with, for example, FKBP or a derivative thereof Fusion to destabilize one or more essential proteins. These fusion proteins can be stabilized by the presence of stabilizers such as Shield-1. As used herein, "rdCMV" refers to a conditional replication-defective cell giant virus.

In a preferred embodiment, the immune response induced by the replication-defective virus is identical or substantially similar in degree and/or magnitude to the corresponding portion of the live virus. In other embodiments, the morphology of the replication-defective virus achieved by electron microscopy analysis is indistinguishable or substantially similar to the corresponding portion of the live virus.

As used herein, "FKBP" refers to the destabilizing protein of SEQ ID NO:11. Fusion proteins containing FKBP are degraded by host cell machinery. As used herein, "FKBP derivative" refers to an FKBP protein or portion thereof that has been altered, deleted, and/or altered by one or more amino acids. The FKBP derivative retains substantially all of the destabilizing properties of FKBP when fused to the protein and also retains substantially all of the ability of FKBP to be stabilized by Shield-1. Preferably, the FKBP derivative has one or more of the following substitutions at the position of the amino acid shown: F15S, V24A, H25R, F36V, E60G, M66T, R71G, D100G, D100N, E102G, K105I and L106P. A FKBP derivative (SEQ ID NO: 12) having a F36V and L106P substitution is particularly preferred. In a preferred embodiment, the nucleic acid encoding a FKBP or FKBP derivative contains at least some codons that are not commonly used in humans for endogenous FKBP. This may reduce the likelihood that the FKBP or FKBP derivative of the fusion protein will rearrange or recombine with its corresponding portion in the human genome. The nucleic acid sequence SEQ ID NO: 13 encodes SEID NO: 12 using these codons.

The term "Shield-1" or "Shld 1" as used herein refers to a synthetic small molecule that binds to wild-type FKBP and its derivatives and acts as a stabilizer. The binding to the F36V derivative is approximately 1,000-fold compared to wild-type FKBP (Clackson et al., 1998, PNAS 95: 10437-42). Shield-1 can be synthesized (essentially as described in the following literature: Holt et al, 1993, J. Am. Chem. Soc. 115: 9925-38 and Yang et al, 2000, J. Med. Chem. 43: 1135-42 and Grimley et al., 2008, Bioorganic & Medicinal Chemistry Letters 18: 759) or purchased from Cheminpharma LLC (Farmington, CT) or Clontech Laboratories (Mountain View, CA). Salts of Shield-1 can also be used in the process of the invention. Shield-1 has the following structure:

The term "fusion" or "fusion protein" as used herein, refers to two polypeptides that are arranged within the framework as part of the same contiguous amino acid sequence. The fusion may be direct such that no additional amino acid residues are present between the polypeptides; or indirectly, such that a small amino acid linker is present to improve performance or increase functionality. In the preferred embodiment, the fusion is straightforward.

The term "pentameric gH complex" or "gH complex" as used herein refers to a complex of five viral proteins on the surface of CMV virions. The complex consists of proteins encoded by UL128, UL130 and UL131 assembled onto gH/gL scaffolds (Wang and Shenk, 2005 Proc Natl Acad Sci) USA. 102: 1815; Ryckman et al., 2008 J. Virol. 82: 60). The sequence of the complex protein from the CMV strain AD 169 is shown by the following GenBank accession numbers: NP_783797.1 (UL128), NP_040067 (UL130), CAA35294.1 (UL131), NP_040009 (gH, also known as UL75), and NP_783793 (gL) , also known as UL115). Some attenuated CMV strains have one or more mutations in UL131 such that the protein is not expressed and thus does not form a gH complex. In such cases, UL131 (using methods such as those in Wang and Shenk, 2005 J. Virol. 79: 10330) should be repaired such that the gH complex is expressed in the rdCMV of the present invention. The virus of the present invention represents a virus that constitutes a pentameric gH complex and assembles five proteins of a pentameric gH complex on the viral envelope.

As used herein, "essential protein" refers to the viral proteins required for viral replication in vivo and in tissue culture. Examples of essential proteins in CMV include, but are not limited to, IE1/2, UL37x1, UL44, UL51, UL52, UL53, UL56, UL77, UL79, UL84, UL87, and UL105.

As used herein, "de-stabilizing essential protein" refers to an essential protein that is expressed and performs its function in viral replication and is degraded in the absence of a stabilizer. In a preferred embodiment, the essential protein is fused to a destabilizing protein such as FKBP or a derivative thereof. Under normal growth conditions (ie, in the absence of a stabilizer), the fusion protein will behave but degraded by host cell machinery. Degradation does not allow essential proteins to play a role in viral replication, thus functionally eliminating essential proteins. In the presence of a stabilizer such as Shield-1, the fusion protein is stable and can perform its function by maintaining the amount of viral replication which is preferably at least 75% of the amount of replication of the CMV that does not contain the essential protein. 80%, 90%, 95%, 99% or 100%.

Replication defective CMV

The method of the invention uses a replication defective CMV (rdCMV) that exhibits a pentameric gH complex. Any attenuated CMV virus that exhibits a pentameric gH complex can be made replication defective according to the methods of the invention. In one embodiment, the attenuated CMV has restored AD 169 of gH complex expression by repairing mutations in the UL131 gene (see Example 1).

A conditional replication-deficient virus is one in which one or more essential viral proteins have been stabilized by a corresponding protein to stabilize the corresponding partial replacement. The destabilizing corresponding portion is encoded by a nucleic acid encoding a fusion protein between the essential protein and the destabilizing protein. Destabilizing essential proteins can only function in the presence of stabilizers to support viral replication. In a preferred embodiment, the method described in U.S. Patent Publication No. 2009/0215169 is used to impart a conditional replication defective phenotype to CMVs that exhibit pentameric gH complexes. Briefly, one or more proteins necessary for CMV replication are fused to a destabilizing protein FKBP or FKBP derivative. Nucleic acids encoding wild type essential proteins are no longer present in rdCMV. The fusion protein is stable and the essential protein acts to support viral replication in the presence of an exogenously added cell-permeable small molecule stabilizer Shield-1 (Shld-1). The replication of rdCMV in the presence of a stabilizer is preferably at least 75%, 80%, 90%, 95% of the amount of replication of the CMV that does not contain the destabilizing fusion protein (eg, the parental decaying CMV used to construct rdCMV). 99% or 100%. In the absence of Shield-1, the destabilizing protein of the fusion protein directs the fusion protein to be substantially degraded by the host cell machinery. CMV cannot be replicated in a certain amount in the absence of essential protein or in the presence of a minimum amount of essential protein CMV infection is produced or maintained in the patient. The replication of rdCMV in the absence of a stabilizer does not occur or is preferably greater than 50%, 75%, 90% compared to CMVs that do not contain a destabilizing fusion protein (eg, the parental attenuated CMV used to construct rdCMV). , 95% or 99%.

A nucleic acid encoding an essential protein for CMV replication and/or establishing/maintaining CMV infection is attached to a nucleic acid encoding FKBP or a derivative thereof using recombinant DNA methods well known in the art. The encoded fusion protein comprises a FKBP or FKBP derivative fused to the essential protein within the framework. The encoded fusion protein is stable in the presence of Shield-1. However, the encoded fusion protein was stable and directed disrupted in the absence of Shield-1. In a preferred embodiment, FKBP is SEQ ID NO:11. In other embodiments, the FKBP derivative comprises one or more FKBPs selected from the group consisting of amino acids: F15S, V24A, H25R, F36V, E60G, M66T, R71G, D100G, D100N, E102G, K105I and L106P. In a more preferred embodiment, the FKBP derivative comprises a F36V and/or L106P substitution (SEQ ID NO: 12). In a more preferred embodiment, the FKBP derivative is encoded by SEQ ID NO:13.

Orientation of essential proteins destabilized by fusion with FKBP or a derivative thereof 1) is required for viral replication; 2) can be adapted to destabilize protein fusion without substantially disrupting the function of the essential protein; and 3) can be adapted to encode A nucleic acid encoding FKBP or a derivative thereof is inserted at the 5' or 3' end of the viral ORF of the protein without substantially destroying the ORF of other surrounding viral genes. In a preferred embodiment, the possibility that the essential protein destabilized by fusion with FBBP or a derivative thereof encodes a non-structural protein and thus is encapsulated into a recombinant CMV virion is reduced. Table 1 shows the CMV genes that meet the above criteria.

The invention encompasses rdCMV comprising a fusion protein having an essential protein fused to a destabilizing protein or a derivative thereof. The essential protein derivative contains one or more amino acid substitutions, additions and/or deletions relative to the wild type essential protein, but still provides the essential protein at least sufficient to support viral replication in the presence of Shield-1. Examples of measuring viral activity are provided in the examples below. Differences between the essential proteins and derivatives of the CMV of interest can be determined using methods known in the art. In one embodiment, sequence consistency is used to determine correlation. The derivatives of the invention will preferably be at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, at least 99% identical to the base sequence. The percent identity is defined as the number of consistent residues divided by the total number of residues and multiplied by 100. If the sequences in the alignment have different lengths (due to the gap) Or the extension, the length of the longest sequence representing the total length value will be used in the calculation.

In some embodiments, one or more of the directed destabilizing viral proteins necessary for viral replication are selected from the group consisting of: IE1/2, UL51, UL52, UL84, UL79, UL87, UL37x1, UL77, and UL53 or derivatives thereof Things. In a particular embodiment, one or more of the directed destabilizing viral proteins necessary for viral replication are selected from the group consisting of IE1/2, UL51, UL52, UL84, UL79, UL87. In a more specific embodiment, one or more of the directed destabilizing viral proteins necessary for viral replication are selected from the group consisting of IE1/2, UL51, UL52, UL79, and UL84.

More than one essential protein can be destabilized by fusion with FKBP or a derivative thereof. In some embodiments, the essential proteins act at different stages of CMV replication and/or infection, including, but not limited to, immediate early, early, or late. In a preferred embodiment, the combination of directed destabilizing viral proteins necessary for viral replication is selected from the group consisting of IE1/2 and UL51, IE1/2 and UL52, IE1/2 and UL79, IE1/2 With UL84, UL84 and UL51, and UL84 and UL52. In a more preferred embodiment, IE1/2 and UL51 are oriented destabilized in the same recombinant CMV. In a preferred embodiment, the fusion protein comprising IE1/2 is SEQ ID NO: 1 and the fusion protein comprising UL51 is SEQ ID NO: 3. SEQ ID NOS: 1 and 3 can be encoded by SEQ ID Nos: 2 and 4, respectively. The genome of rdCMV with destabilized IE 1/2 and UL51 is shown in SEQ ID NO: 14.

FKBP or a derivative thereof can be directly or indirectly fused to an essential protein. In a preferred embodiment, FKBP or a derivative thereof is directly fused to an essential protein.

FKBP or a derivative thereof can be fused to an essential protein at the N or C terminus of an essential protein. In a preferred embodiment, the FKBP line is fused to the N-terminus of the essential protein.

More than one FKBP or a derivative thereof can be fused to an essential protein. In embodiments in which more than one FKBP or derivative thereof is fused to an essential protein, each FKBP or derivative thereof may be the same or different. In a preferred embodiment, a FKBP or derivative thereof is fused to an essential protein.

Other inactivation methods

In some embodiments, the rdCMV described above is further inactivated using chemical or physical inactivation. Examples of such methods include heat treatment, incubation with formaldehyde, beta-propiolactone (BPL) or diethyleneimine (BEI), or gamma irradiation. Preferred methods do not destroy or substantially destroy immunogenicity, including, but not limited to, immunogenicity induced by the pentameric gH complex. Thus, the CMV-induced immune response is maintained or substantially maintained as compared to rdCMV without other inactivation treatment. In a preferred embodiment, the ability of the further inactivated CMV to induce neutralizing antibodies is comparable to those of the rdCMV inducers treated by no other inactivation treatment. The immunogenicity of CMV, including CMV (including pentameric gH complex), is determined empirically by an inactivation scheme by either or both of chemical or physical methods.

Evaluation of viral replication

Those skilled in the art can use viral replication assays to determine the utility of a particular essential protein fused to FKBP or a derivative thereof. Due to the attachment of FKBP or its derivatives to essential proteins in the presence of Shield-1 Qualitatively affects gene expression/coding product function, therefore rdCMV should be in a ratio comparable to parental CMV in the presence of Shield-1 (preferably at least 75%, 80%, 90%, 95% of the amount of parental virus, 99% or 100%) copy. In the absence of Shieid-1, replication of rdCMV is substantially different from that of parental CMV (reduced by more than 50%, 75%, 90%, 95%, 99% or 100 compared to CMV without destabilizing fusion protein) %).

In a preferred embodiment, the rdCMV replicates preferably at least 90%, more preferably at least 95%, and most preferably at least 99% of the non-rdCMV replication in the presence of at least 2 μM Shield-1.

In one embodiment, the viral titer comprising the composition of the rdCMV of the invention is at least 10 ⁵ pfu/ml, more preferably at least 10 ⁷ pfu/ml, in the presence of at least 2 μM Shield-1.

Conversely, in the absence of Shield-1, rdCMV should not be replicated in nature. The quality of the replication defective mechanism is judged by the degree of rigor of control under conditions that do not allow for viral replication (i.e., the infectious virion of the progeny virions under such conditions). In the absence of Shield-1, the rdCMV of the invention is substantially incapable of replication (in cell culture or within a patient). Its replication in ARPE-19 cells and other types of human primary cells is conditional and requires a molar concentration of Shield-1 in the culture medium greater than 0.1 μM, preferably at least 2 μM to maintain viral replication.

In one embodiment, the composition comprising the rdCMV of the invention has a virus titer of less than 2 pfu/ml, more preferably less than 1 pfu/ml, in the absence of Shield-1.

The method of evaluating CMV replication can be used to evaluate the absence of Shield-1 or There is rdCMV replication underneath. However, in the preferred embodiment, TCID 50 is used.

In another embodiment, the rdCMV titer is determined by a 50% tissue culture infectious dose (TCID50) assay. Briefly, this dilution analysis quantifies the amount of virus required to kill 50% of infected hosts. Host cells (eg, ARPE-19 cells) are plated and serial dilutions of the virus are added. After incubation, the percentage of cell death (i.e., infected cells) for each virus dilution was observed and recorded. Use the results to mathematically calculate TCID50.

In another embodiment, the rdCMV titer is determined using plaque assay. Viral plaque assay determines the number of plaque forming units (pfu) in the virus sample. Briefly, a confluent monolayer of host cells (eg, ARPE-19 cells) is infected with different dilutions of rdCMV and covered with semi-solid medium (eg, agar or carboxymethylcellulose) to prevent viral infection from spreading arbitrarily . Viral plaques are formed when the virus infects cells in a fixed cell monolayer. The virus-infected cells will dissolve and spread the infection to adjacent cells where the infection-dissolution cycle is repeated. The infected cell area will produce plaques (infected areas surrounded by uninfected cells) which can be visually observed or observed using an optical microscope. The number of plaque forming units per sample unit volume (pfu/mL) was calculated by counting the plaques and using the results in combination with the dilution factors used to prepare the plates. The pfu/mL results represent the number of infectious particles in the sample and are based on the assumption that each plaque formed represents an infected virus particle.

In another embodiment, the hu-SCID mouse model is used to assess the ability of rdCMV to replicate in vivo. Briefly, several human fetal tissues, such as the thymus and liver, are surgically implanted into the renal capsule of SCID mice. in After 2 to 3 months, when human tissue is vascularized, rdCMV is inoculated. Viral titers were evaluated in plaque assays 3 to 4 weeks after inoculation. Animal experiments can be performed in the absence or presence of Shield-1.

Assessment of immune response

Administration of the rdCMV of the invention induces an immune response to CMV, preferably a protective immune response, which can treat a patient's CMV infection or a condition associated with the infection and/or reduce its likelihood. The immune response is at least partially directed to the pentameric gH complex.

The immune response induced by rdCMV can be assessed using methods known in the art.

Animal models known in the art can be used to evaluate the protective effects of administration of rdCMV. In one embodiment, the immune sera neutralization ability of an individual administered rdCMV can be analyzed, including but not limited to blocking of the virus or entry into the host cell. In other embodiments, the T cells of an individual administered with rdCMV can be analyzed for the ability to produce interleukins in the presence of the antigen of interest, including but not limited to, interferon gamma. An animal stimulation model can also be used to determine the immunologically effective amount of the immunogen.

Viral neutralization refers to the ability of a virus-specific antibody to disrupt viral entry and/or replication in culture. A common assay for measuring neutralizing activity is viral plaque reduction assay. Neutralization assays in the present invention refer to serum titration that blocks the entry of a virus into a cell. The NT50 titer is defined as the reciprocal of the serum dilution that blocks 50% of the input virus in the virus neutralization assay. The NT50 titer was obtained from a nonlinear logic four-parameter curve fit.

Manufacture of replication defective CMV

The invention encompasses methods of making rdCMV. The rdCMV of the present invention is propagated on epithelial cells, preferably human epithelial cells, and more preferably human retinal pigment epithelial cells in the presence of a stabilizer such as Shield-1. In other embodiments, the human retinal pigment epithelial cell line is deposited with ARPE-19 cells of the American Type Culture Collection (ATCC) under accession number CRL-2302. In some embodiments, the Shield-1 is present in the tissue culture medium at a concentration of at least 0.5 μM. In a preferred embodiment, the Shield-1 is present in the tissue culture medium at a concentration of at least 2.0 μM.

In some embodiments, the cell line used to propagate rdCMV is grown on a microcarrier. Microcarriers are support matrices that allow adherent cells to grow in a rotating flask or bioreactor, such as a rotating wall microgravity bioreactor and a fluidized bed bioreactor. Microcarriers are typically 125-250 μM spheres, the density of which allows them to be maintained in suspension while gently agitating. Microcarriers can be made from a variety of different materials including, but not limited to, DEAE-dextran, glass, polystyrene plastic, acrylamide, and collagen. Microcarriers can have different surface components including, but not limited to, extracellular matrix proteins, recombinant proteins, peptides, and charged molecules. Other high-density cell culture systems such as Corning HyperFlask® and HyperStack® systems can also be used.

Cell-free tissue culture medium can be harvested and rdCMV can be purified therefrom. The CMV virions are about 200 nm in diameter and can be separated from other proteins present in the harvested medium using techniques known in the art including, but not limited to, via a density gradient or a 20% sorbitol mat ( Ultracentrifugation of Sorbitol cushion). The amount of protein in the vaccine can be determined by Bradford analysis.

Shield-1 can be used in conjunction with FKBP to control the replication of rdCMV. On completion After the desired amount of virus in the tissue culture cells is propagated, the ability to replicate is no longer satisfactory. Shield-1 is withdrawn from rdCMV to replicate the virus as defective (eg, to the patient). In one embodiment, rdCMV is purified from Shield-1 by washing one or more times. In another embodiment, rdCMV is purified from Shield-1 via ultracentrifugation. In another embodiment, rdCMV is purified from Shield-1 via diafiltration. Diafiltration is typically used to purify viral particles. In one embodiment, a filter having a pore size of about 750 kilodaltons is used, which will only allow Shield-1 to pass through the aperture.

After purification of rdCMV from Shield-1, a small amount of residual Shield-1 remaining in the rdCMV composition may be present. In one embodiment, the amount of Shld-1 in the CMV composition after purification is maintained at up to 1/100 of the amount required for replication in tissue culture. In another embodiment, the amount of Shield-1 in the rdCMV composition is 0.1 μM or less after purification. In another embodiment, the amount of Shield-1 in the rdCMV composition is undetectable after purification.

The determination of the amount of Shield-1 in the composition can be detected using LC/MS (liquid chromatography-mass spectrometry) or HPLC/MS (high performance liquid chromatography-mass spectrometry) analysis. These techniques combine the physical separation capabilities and mass analysis capabilities of LC or HPLC and detect the chemicals of interest in the composite mixture.

Adjuvant

An adjuvant is a substance that aids in the immune response of an immunogen. The adjuvant may act by different mechanisms such as one or more of: extending antigenic biology or immunological half-life; improving antigen delivery to antigen presenting cells; improving antigen processing and presentation by antigen presenting cells; Dose savings and An immunomodulatory interleukin is induced (Vogel, 2000, Clin Infect Dis 30: S266). In some embodiments, the compositions of the invention comprise rdCMV and an adjuvant.

A variety of different types of adjuvants can be used to aid in the production of an immune response. Examples of specific adjuvants include aluminum hydroxide; aluminum phosphate, aluminum hydroxyphosphate, amorphous aluminum hydroxyphosphate sulfate adjuvant (AAHSA) or other aluminum salts; calcium phosphate; DNA CpG motif; monophosphonium lipid A; cholera Toxin; E. coli heat labile toxin; pertussis toxin; cell wall 醯 dipeptide; Freund's incomplete adjuvant; MF59; SAF; immunostimulating complex; liposome; biodegradable Microspheres; saponins; non-ionic blocking copolymers; cell wall purine peptide analogs; polyphosphazenes; synthetic polynucleotides; IFN-γ; IL-2; IL-12; (Vogel, 2000, Clin Infect Dis 30: S266; Klein et al, 2000, J Pharm Sci 89: 311; Rimmelzwaan et al, 2001, Vaccine 19: 1180; Kersten, 2003, Vaccine 21: 915; O'Hagen, 2001 , Curr.Drug Target Infect.Disord.1:273).

In some embodiments, oil-based adjuvants are used in the compositions of the invention, including but not limited to incomplete Freund's adjuvant and MF59.

In other embodiments, microparticle adjuvants are used in the compositions of the invention, including but not limited to, ISCOMATRIX® adjuvants and/or aluminum phosphate adjuvants.

Pharmaceutical composition

Another feature of the invention is the use of a recombinant CMV as described herein in a composition, preferably an immunogenic composition or vaccine, for use in treating a patient having a CMV infection and/or reducing the likelihood of CMV infection Sex. suitable Preferably, the composition comprises a pharmaceutically acceptable carrier.

"Pharmaceutically acceptable carrier" means a liquid filler, diluent or encapsulating material that can be safely administered for systemic administration. Depending on the particular route of administration, a variety of pharmaceutically acceptable carriers well known in the art can be used. The carriers may be selected from the group consisting of sugars, starches, celluloses and derivatives thereof, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffer solutions. (including phosphate buffered saline), emulsifier, isotonic saline and non-pyrogenic water. In particular, pharmaceutically acceptable carriers may contain different components such as buffers, sterile water for injection, physiological saline or phosphate buffered saline, sucrose, histidine, salts and polysorbates. Terms such as "physiologically acceptable", "diluent" or "excipient" are used interchangeably.

Vaccine formulation procedures are disclosed, for example, in New Generation Vaccines (1997, Levine et al, Marcel Dekker, Inc., New York, Basel, Hong Kong), which is incorporated herein by reference.

Formulation

In some embodiments, a rdCMV of the invention is administered to a patient to elicit an immune response. It is desirable to minimize or avoid loss of rdCMV composition efficacy during storage of the immunogenic composition. Conditions that support this purpose include (but are not limited to): (1) sustained storage stability, (2) tolerance to the freeze-thaw cycle, (3) stability at ambient temperature for up to one week, and (4) maintenance of the immunogen Sex, (5) compatible with adjuvant strategies. Conditions that affect the stability of rdCMV include, but are not limited to, buffer pH, buffer ionic strength, presence/absence of specific excipients, and temperature. The composition contains a buffer to increase the suitability as an epidemic The stability of the purified rdCMV virus particles of the seedling composition.

The maintenance of viral particle integrity can be assessed by immunogenicity analysis in mouse and/or virus entry assays. The viral entry event depends on the integrity and function of the viral glycoprotein including the pentameric gH complex. The pentameric gH complex also provides substantial immunogenicity of rdCMV, thereby correlating the two properties.

In some embodiments, rdCMV is stored in a buffer (pH between 5 and 7) comprising 15-35 mM histidine and 100-200 mM NaCl. In a more specific embodiment, the buffer comprises 25 mM histidine and 150 mM NaCl (pH 6).

In other embodiments, sugars may be added to provide further stability, such as polyols (including but not limited to mannitol and sorbitol); monoalcohols including, but not limited to, glucose, mannose, galactose, and fructose Glycols (including but not limited to lactose, maltose, sucrose, lactulose and trehalose) and trisaccharides (including but not limited to, raffinose and melezitose). In a more specific embodiment, the sugar is sucrose. In an even more specific embodiment, the sucrose is between 5% and 15%.

In a preferred embodiment, rdCMV is stored in a buffer comprising 25 mM histidine, 150 mM NaCl, 9% sucrose (pH 6).

Cast

The rdCMV described herein can be formulated and administered to a patient using the guidance provided herein in conjunction with well known techniques in the art. Guidance for the administration of pharmaceuticals is generally provided, for example, in the following literature: Vaccines , ed. Plotkin and Orenstein, WBSanders, 1999; Remington's Pharmaceutical Sciences 20th edition, edited by Gennaro, Mack Publishing, 2000; and Modem Pharmaceutics 2nd edition, Edit Banker and Rhodes, Marcel Dekker, 1990.

Vaccines can be administered by different routes such as subcutaneous, intramuscular, intravenous, mucosal, parenteral, transdermal or intradermal. Subcutaneous and intramuscular administration can be performed using, for example, a needle or a jet injector. In one embodiment, the vaccine of the invention is administered intramuscularly. Transdermal or intradermal delivery can be achieved via intradermal syringe needle injection or enabling devices such as microneedle or microarray patches.

The compositions described herein can be administered in a manner compatible with the dosage formulation and in an amount effective to treat CMV infection (including primary, recurrent, and/or re-) and/or reduce the likelihood of immunogenicity. . In the context of the present invention, the dosage administered to the patient should be sufficient to effect a beneficial response in the patient over time, such as reducing the extent of CMV infection, improving the symptoms of the disease associated with CMV infection and/or shortening the duration and/or severity of CMV infection. Or reduce the likelihood of CMV infection (including primary, recurrent, and/or re-).

Suitable dosing regimens can be readily determined by those skilled in the art and are preferably determined by well-known factors in the industry, including the age, weight, sex, and medical condition of the patient; the route of administration; the desired effect; and the particular combination employed. Things. In determining an effective amount of rdCMV to be administered in the treatment or prevention against CMV, the physician can assess circulating plasma concentrations of the virus, disease progression, and/or production of anti-CMV antibodies. The dosage of the vaccine composition consists of a range of 10 ³ to 10 ¹² plaque forming units (pfu). In various embodiments, the dosage range is from 10 ⁴ to 10 ¹⁰ pfu, from 10 ⁵ to 10 ⁹ pfu, from 10 ⁶ to 10 ⁸ pfu, or any of these ranges. When more than one vaccine (i.e., in combination vaccine) is to be administered, the amount of each vaccine agent is within its stated range.

The vaccine composition can be administered in a single dose or multiple dose mode. The vaccine can be prepared with an adjuvant several hours or days prior to administration, identification of a stabilizing buffer, and a suitable adjuvant composition. Vaccines can be administered in the usual practice volume (in the range of 0.1 mL to 0.5 mL).

Dose timing depends on factors well known in the art. After initial administration, one or more additional doses may be administered to maintain and/or enhance antibody titer and T cell immunity. Additional enhancement may be required to maintain the degree of protection of the immune response, as reflected by antibody titers and T cell immunity (eg, ELISPOT). The extent of these immune responses is the subject of clinical research.

For combination vaccination, each immunogen can be administered together in one composition or separately in different compositions. The rdCMV lines described herein are administered concurrently with one or more desired immunogens. The term "simultaneously" is not limited to the administration of a therapeutic agent at exactly the same time, but rather means that the rdCMV and other desired immunogenic lines described herein are administered to the individual in a sequence and at intervals of time such that they are One works to provide an added benefit compared to when it is otherwise administered. For example, each therapeutic agent can be administered sequentially at the same time or in any order at different time points; however, if not administered at the same time, it should be sufficiently close in time to provide the desired therapeutic effect. Each therapeutic agent can be administered separately, in any suitable form, and by any suitable route.

Patient population

"Patient" means a mammal that can be infected with CMV. In a preferred embodiment, the patient is a human. Patients can be treated prophylactically or therapeutically. Preventive Treatment provides sufficient protective immunity to reduce the likelihood or severity of CMV infection, including primary infections, recurrent infections (ie, those from latent CMV reactivation) and reinfection (ie, they Originated from an infected person with a different strain of CMV than the patient previously experienced). Therapeutic treatment can be performed to reduce the severity of CMV infection or to reduce the likelihood/severity of relapse or reinfection.

Treatment can be performed using a pharmaceutical composition comprising rdCMV as described herein. Pharmaceutical compositions can be administered to a general population, particularly those with an increased risk of CMV infection (primary, recurrent, or re-) or CMV infections (e.g., immunocompromised individuals, transplant patients, or pregnant women). In one embodiment, women of childbearing age, particularly early adolescent women, are vaccinated to reduce the likelihood of CMV infection (primary, relapsing or re-) during pregnancy.

Those who need treatment include those who have already become infected, and those who prefer to be infected or who are expected to reduce the likelihood of infection. Treatment may ameliorate the symptoms of the disease associated with CMV infection and/or shorten the duration and/or severity of the CMV infection, including infections caused by latent CMV reactivation.

Patients at increased risk of CMV infection (primary, recurrent, or re-) include immunocompromised patients or patients facing treatments that cause reduced immunity (eg, undergoing chemotherapy or radiation therapy for cancer or taking immunosuppressive drugs). As used herein, "immune immunity" refers to an immune system that is less resistant to infection by an immune response that does not function properly or that does not function as a normal healthy adult. Examples of patients with reduced immunity are infants, young children, elderly people, pregnant women, or patients with diseases that affect the function of the immune system (such as HIV infection or AIDS).

Instance

Examples are provided below to further illustrate various features of the invention. These examples also illustrate useful methods of practicing the invention. These examples do not limit the claimed invention.

Example 1: Recovery of pentameric gH complex

Infectious CMV bacterial artificial chromosomes were constructed such that the coding virions exhibited a pentameric gH complex consisting of UL128, UL130 and UL131 assembled onto a gH/gL scaffold.

The CMV strain AD 169 strain was originally isolated from the proliferating gland of a 7 year old girl (Elek and Stern, 1974, Lancet, 1:1). The virus was passaged 58 times in several types of human fibroblasts to attenuate the virus (Neff et al., 1979, Proc Soc Exp Biol Med, 160:32), with the last 5 passages in WI-38 human fibers. Performed in mother cells. This passaged variant of AD 169 virus (referred to as Merck AD 169, MAD 169 in this study) was used as a parental virus to construct an infectious BAC line. Both the parental virus AD 169 and the passaged variant virus MAD 169 do not exhibit a UL131 or pentameric gH complex.

The infectious bacteria artificial chromosome (BAC) pure line was constructed using MAD 169 as a parental virus. The BAC vector is a molecular tool that allows genetic manipulation of large size DNA fragments in E. coli, such as the CMV genome (about 230 Kb). The BAC element and the GFP marker gene (between US28 and US29 ORF in the viral genome) were inserted immediately following the stop codon of the US28 open reading frame, wherein LoxP sites were established at both ends of the fragment (Fig. 1A). Briefly, the DNA fragment containing the GFP expression cassette and the CMV US28-US29 sequence, which are flanked by two loxP sites, were synthesized and cloned into pBeloBAC11 In the body. The BAC vector was linearized with restriction enzyme Pme I and co-transfected into MRC-5 cells with MAD 169 DNA extracted from purified virions. The recombinant system identified by green fluorescent expression was purified by plaque. After one round of amplification, the viral genome in circular form was extracted from infected cells and electroporated into E. coli DH10 cells. The presence of US28 and US29 regions in bacterial colonies was screened by PCR. Candidate lines were further tested by EcoR I, EcoR V, Hind III, Spe I and Bam HI restriction assays. After screening, one pure line of bMAD-GFP showed the same restriction pattern as the parental MAD 169 virus.

The frameshift mutation in the first exon of UL131 that causes epithelial deficiencies in MAD 169 was genetically repaired in E. coli (Fig. IB). Specifically, one of the 7 adenine nucleotides in the UL131 gene was deleted and one nt A was absent (Fig. 1B). A 1 nt deletion is sufficient to rescue epithelial and endothelial cell tropism due to UL131 and (and thus) pentameric gH complexes. Performance was confirmed by ELISA and western blot (data not shown). This pure line was further modified by LoxP/Cre recombination to remove the BAC segment. BAC DNA was transfected into ARPE-19 cells, human retinal pigment epithelial cells (ATCC Accession No. CRL-2302) to recover infectious virus (Fig. 1C). The resulting infectious virus is referred to as the BAC-derived epithelial MAD 169 virus (beMAD), which differs from MAD 169 only in the following two loci: (1) the UL131 ORF in which a single adenine nucleotide is deleted; 2) A 34 bp LoxP site inserted between US28 and US29 ORF (see Table 2).

The genome of the BAC pure beMAD is completely sequenced. The overall basis of beMAD Since the group structure is identical to that reported by the ATCC AD 169 variant (GenBank Accession No. X17403), the variant includes two distinct regions, the Long Independent Region (UL) and the Short Unique Region (US). Each unique region includes two repeating sequences, namely long-end repeat (TRL)-long internal repeat (IRL), short-end repeat (TRS)-short internal repeat (IRS). The growth kinetics of the passaged variants MAD 169 and beMAD-derived viruses were indistinguishable in the human fibroblast cell line MRC-5 cells (ATCC Accession No. CCL-171) (data not shown). Since no gH complex is required for growth on fibroblast cells, the difference in gH complex performance between MAD 169 and beMAD is not relevant.

Example 2: Effect of conventional inactivation method on gH complex

The effects of two conventional virus inactivation methods (gamma-irradiation and beta-propiolactone (BPL)) on CMV expressing gH were investigated.

Gamma-irradiation was performed on the lyophilized virions. Concentration of 0.15 mg/mL in HNS (25 mM histidine) using a conservative lyophilization cycle (freezing at -50 °C and initial drying at -35 °C for about 30 hr followed by secondary drying at 25 °C for 6 hr) The recombinant CMV vaccine formulation in 150 mM NaCl, 9% w/v sucrose, pH 6.0) was lyophilized to obtain a dry powder. Freeze the vaccine in a 3 mL glass vial Dry, each of which is filled with 0.5 ml. At the end of lyophilization, the vials were stoppered in a nitrogen atmosphere and the samples removed, labeled, crimped and stored at -70 °C until gamma irradiation. The vial was irradiated with the desired irradiation dose under a Co irradiator.

For BPL treatment, BPL stock was added to the crude virus culture supernatant grown on ARPE-19 cells to achieve a final concentration of 0.01% or 0.1% (v/v). The reaction was terminated with sodium thiosulfate at various time points. The BPL-treated CMV-expressing CMV was then purified by ultracentrifugation.

The inactivation kinetics of the two methods were determined by plaque assay in ARPE-19 cells. Briefly, serial dilutions of virus samples in PBS were prepared and 0.1 mL was used to inoculate wells of 6-well plates in which ARPE-19 cells were seeded. The plates were incubated for 1 hr at 37 ° C, followed by the addition of 6 mL of 0.5% agarose overlying medium per well. The plates were incubated for 18 days at 37 °C. To visualize plaques, approximately 0.5 mL of 5 mg/mL MTT solution (thiazole blue tetrazolium bromide, Sigma M5655) was added to each well. Plates were incubated for 2-4 hr at 37 °C and plaques were counted under lightbox (Figures 2A and 2C).

The immunogenicity of the inactive CMV expressing gH was analyzed by measuring the induction of neutralizing antibody titers in mice. Briefly, female Balb/c mice (n=10) were immunized with 2.5 μg CMV/dose at weeks 0 and 3. Serum was collected at week 4 and assessed for neutralizing activity against viral epithelium. Neutralization titer (NT50) is defined as the reciprocal of serum dilution that reduces viral epithelial entry by 50% compared to the negative control. The results from mouse immunogenicity studies showed that two conventional inactivation methods had a negative effect on the induction of neutralizing antibody titers by CMV expressing gH (Figures 2B and 2D). Reduction of NT50 titer and The duration is related by gamma-irradiation or BPL treatment. Prolonged treatment made the CMV expressing the pentameric gH complex more similar to the parental AD 169 CMV in immunogenicity in mice. Similar results were observed when γ-irradiated or BPL-inactivated vaccines were tested in rabbits and rhesus monkeys (data not shown). These observations show that the pentameric gH complex is sensitive to both inactivation methods under the selected inactivation conditions.

Example 3: Construction and screening of FKBP-essential protein fusions

The CMV was constructed using the attenuated AD 169 strain skeleton, which regained its epithelial tropism and was conditionally replicated. The method described in Example 1 was used to restore epithelial tropism.

Viral proteins to be fused to FKBP derivatives are selected based on two criteria. First, the protein of interest was not detected in the CMV virions by proteomic analysis (Varnum et al., 2004, J. Virol. 78: 10960), thereby reducing the possibility of incorporating the FKBP fusion protein into the virus. Second, the protein of interest is critical for viral replication in tissue culture.

The FKBP derivative (SEQ ID NO: 12) was individually fused to 12 essential viral proteins using beMAD as the parental virus, including IE1/2 (SEQ ID NO: 1), pUL37x1, pUL44, pUL51 (SEQ ID NO: 3), pUL52 (SEQ ID NO: 5), pUL53, pUL56, pUL77, pUL79 (SEQ ID NO: 7), pUL84 (SEQ ID NO: 9), pUL87 and pUL105. Also constructed is a virus having two different essential proteins fused to FKBP, which fused each of IE1/2 and UL51 to a FKBP derivative (the genome of rdCMV with destabilized IE1/2 and UL51 is shown in SEQ ID) NO: 14)). After construction, transfect all recombinant BAC DNA to ARPE-19 cells were cultured in medium containing Shld-1.

The dependence of virus growth on Shld-1 was examined. IE1/2, UL51, UL52, UL84, UL79, and UL87 fusion viruses were easily rescued with 2 μM Shld-1 in plaque assays (data not shown). The plaques were also produced by the UL37x 1, UL77 and UL53 viruses, but the plaques were small and they grew significantly slower than the parental beMAD. Increasing the Shld-1 concentration to 10 μM did not significantly promote virus growth (data not shown). The UL56 and UL105 fusions were not restored, indicating that tagging these proteins would disrupt the function of the proteins or the performance of adjacent genes.

Different concentrations of Shld-1 were used in other experiments to further evaluate viral replication in the presence or absence of Shld-1. ARPE-19 cells were infected by CMV expressing gH with an MOI of 0.01 pfu/ml, which also contained FKBP derivatives fused to the essential protein. One hour after infection, the cells were washed twice with fresh medium to remove Shld-1 from the inoculum. The inoculum was then added to ARPE-19 cells cultured in medium containing 0.05 μM, 0.1 μM, 0.5 μM or 2 μM Shield-1. Seven days after infection, the cell-free progeny virus in the supernatant was collected and titrated onto ARPE-19 cells supplemented with 2 mM Shield-1. Viral titers were determined by 50% tissue culture infectious dose (TCID50) analysis. Briefly, this dilution analysis quantifies the amount of virus required to kill 50% of infected hosts. ARPE-19 cells were plated and serial dilutions of the virus were added. After incubation, the percentage of cell death (i.e., infected cells) for each virus dilution was manually observed and recorded. Use the results to mathematically calculate TCID50.

As shown in Figure 3, all valid copies of CMV containing FKBP fusions Depending on the Shield-1 concentration, but to a different extent. Lower concentrations of Shield-1 generally reduce the potency produced by progeny viruses. Of the viruses with a single fusion, only copies of UL51 and UL52 require Shield-1. Other viruses with a single fusion (IE1/2, UL84, UL79, and UL87) can produce detectable progeny viruses in the absence of Shield-1. When FKBP derivatives are fused to UL51 or UL52, the regulation is most stringent.

The growth kinetics of the virus with the IE1/2, UL51, IE1/2-UL51 fusions were compared to the parental beMAD virus in the presence of 2 μM Shld-1 or in the absence of Shld-1. As shown in Figure 4, the growth kinetics of single or double fusions were comparable to the parental beMAD in the presence of Shld-1. However, in the absence of Shld-1, only IE1/2 can replicate, but at a slower and slower rate than the parental beMAD.

The control rigor of viral replication in double fusion viruses was also tested in different cell types (Figure 5). Such cells include human umbilical vein cells (HUVEC), MRC-5 fibroblasts, aortic smooth muscle cells (AoMC), skeletal muscle cells (SKMC), and CCF-STTG1 astrocytoma cells. Cells were infected by IE1/2-UL51 fusion virus (except CCF-STTG1 infected with 5 pfu/cell MOI) with an MOI of 0.01 pfu/cell and subsequently incubated in medium with or without Shield-1. All cell types support soluble viral replication in the presence of Shield-1. No virus production was detected in the absence of Shield-1.

Example 4: Immunogenicity of IE1/2-UL51 double fusion virus in animals

The immunogenicity of the IE1/2-UL51 double fusion virus was evaluated in mice, rabbits and rhesus monkeys. First compare the IE1/2-UL51 double fusion virus in mice or A dose-dependent neutralization reaction of the parental beMAD virus (Fig. 6A). 6-week-old female BALB/c mice were immunized at week 0 and week 4 with beMAD or IE1/2-UL51 double fusion virus at doses ranging from 0.12 μg to 10 μg. Serum samples were taken at week 6 and analyzed on ARPE-19 cells by CMV microneutralization assay as previously described (Tang et al., Vaccine, "A novel throughput neutralization assay for supporting clinical evaluations of human cytomegalovirus vaccines", Published on August 30, 2011 in electronic format, doi: 10.1016/j.vaccine.2011.08.086). The reactions were compared at doses of 0.12 μg, 0.37 μg, 1.1 μg, 3.3 μg, and 10 μg. At low doses (0.12 μg to 1.1 μg), beMAD is slightly more immunogenic and neutralizing antibodies are always detected at doses above 0.37 μg. In the high dose range (3.3 μg and 10 μg), the neutralization antibody titers induced by the two viruses were comparable.

Next, the immunogenicity of different viruses at a dose of 10 μg in rabbits was compared. Female NZW rabbits were immunized with 10 μg of beMAD or the indicated fusion virus at weeks 0, 3 and 8. Serum at week 10 was collected and analyzed on ARPE-19 cells by CMV microneutralization assay (Fig. 5B). beMAD, single fusion virus IE1/2 or UL51 and double fusion virus IE1/2-UL51 significantly induced higher than MAD 169 neutralizing antibody titer, MAD 169 is a virus similar to AD 169 and lacking pentameric gH complex . This confirms that the expression of the gH complex by the virus significantly increases the immunogenicity of the recombinant CMV.

Next, the immunogenicity of 100 μg of the double-fused IE1/2-UL51 virus or the parental beMAD virus was tested in rhesus monkeys. Serum at week 12 was collected and analyzed on ARPE-19 cells by CMV microneutralization assay. Week 12 (3rd time The GMT NT50 titers of the drug are 11,500 or 15600, respectively. This equivalent price is comparable to the NT50 titer observed in naturally infected individuals (Fig. 5C).

The lifespan of the immune response induced by the double fusion virus IE1/2-UL51 CMV vaccine was shown in rhesus monkeys. Animals were vaccinated at 10 μg/dose or 100 μg/dose of double fusion virus IE1/2-UL51 (based on total protein amount). Formulations of 10 μg/dose vaccine with amorphous aluminum hydroxyphosphate sulfate (AAHS) or ISCOMATRIX® adjuvant are also included. Vaccines were administered in rhesus monkeys (n=5) at weeks 0, 8, and 24. For comparison, the control group received 30 μg/dose of recombinant gB formulated with MF59 adjuvant at week 0, week 4, and week 24. The geometric mean (GMT) of the reciprocal of NT50 titers for all groups is presented on the vertical axis (Figure 7). Neutralizing antibody titers >40 were not detected in any of the monkeys prior to vaccination. The lowest neutralizing activity was tested for all groups at week 4 after the first dose, neutralizing antibody titers at approximately week 12 and week 28 (4 weeks after the second and third vaccinations, respectively) Reach the peak. The peak GMT of the 100 μg/dose group at week 28 was 14,500 (approximately 3 times higher than the 4,660 titer of the 10 μg/dose group). The ISCOMATRIX® adjuvant, rather than AAHS, provides adjuvant benefit compared to the 10 μg/dose group. The ISCOMATRIX® group had a GMT of 15,800 at week 28, while the AAHS group was 3,000 and the 10 μg/dose group was 4,660. The lowest neutralizing activity of the control (gB/MF59) group was tested, with a peak GMT never exceeding 200. At the 72nd week of the study, nearly one year after the completion of the vaccination program at weeks 0, 8, and 24, the GMT of the 100 μg/dose group and the ISCOMATRIX® formulation group was maintained at 1400 and 3000, respectively. At this time, the GMT of the 10 μg/dose group and the AAHS group was about 200.

Collected from the 28th week of the vaccination program (4 weeks after the third dose) from Heng Peripheral blood mononuclear cells (PBMC) were evaluated and evaluated in the IFN-γ ELISPOT assay. The monkey was vaccinated with a double fusion virus IE1/2-UL51 at 100 μg/dose (Fig. 8A) or 10 μg/dose (Fig. 8B-8D). In addition, 10 μg/dose was dispensed without adjuvant (Fig. 8B) or with AAHS (Fig. 8C) or ISCOMATRIX® (Fig. 8D) adjuvant. Ex vivo production of IFN-[gamma] was stimulated by pooling overlying peptide antigens representing five HCMV antigens. The HCMV antigens used were IE1 and IE2 (two viral regulatory proteins) and pp65, gB and pp 150 (major viral structural antigens). The quality of T cell responses was assessed by the magnitude of the ELISPOT reaction (geometric mean) and the ratio of responders to viral antigens. There was no antigen-specific ELISPOT titer in any of the monkeys prior to vaccination (data not shown).

At week 28, the geometric mean of the ELISPOT responses to the five HCMV antigens (ie, IE1, IE2, pp65, gB, and pp150) were 186, 132, 253, 87, and 257 spotted cells, respectively. SFC)/10 ⁶ PBMCs (for 100 μg/dose group) versus 21, 24, 107, 111, 33 SFC/10 ⁶ PBMCs (for 10 μg/dose group) 8A and 8B). Responders in each group (n=5) were scored based on a cut-off criterion of more than 55 SFC/10 ⁶ PBMCs and an antigen-specific reaction that was more than 3-fold higher than the dimethyl hydrazine (DMSO) reaction. The number of responders to the five HCMV antigens (ie, IE1, IE2, pp65, gB, and pp 150) was 4, 4, 5, 1, and 3 (for the 100 μg/dose group) versus 1 1, 5, 4, 0 (for the 10 μg/dose group).

The effect of ISCOMATRIX® adjuvant on T cell responses to 10 μg/dose double fusion virus IE1/2-UL51 is shown in Figure 8D. The geometric mean values of the ELISPOT reactions for the five HCMV antigens (ie, IE1, IE2, pp65, gB, and pp 150) were 114, 53, 491, 85, 113 SFC/10 ⁶ PBMCs, respectively. The number of responders in the group (n=5) was 3, 2, 5, 3, and 3, respectively. The magnitude and magnitude of T cell responses in the group with ISCOMATRIX® adjuvant were similar to those in the 100 μg/dose group.

After stimulation with HCMV antigen (pp65, IE1, IE2 or whole HCMV virions), further analysis of intracellular interleukin staining with 10 μg/dose or 100 μg/dose with ISCOMATRIX® double fusion virus IE1/2- PBMC of animals vaccinated with UL51 (based on total protein amount). The negative control was an untreated monkey of the unvaccinated double fusion virus IE1/2-UL51, while the positive control was staphylococcal enterotoxin B (SEB). Figure 9 shows that the negative control showed the lowest response to all antigen stimulation but responded to the positive control staphylococcal enterotoxin B (SEB) as expected. All 10 vaccinated monkeys from both groups responded to HCMV-specific antigens in similar magnitudes and patterns. The geometric mean of each of the 10 monkeys for each antigen was calculated. When all of the monkey PBMCs were stimulated with the CMV antigen peptide pool (i.e., pp65, IE1, and IE2), they all showed comparable CD8+ (Fig. 9A) and CD4+ (Fig. 9B) T cell responses; but when stimulated with intact HCMV virions When it is preferentially displayed, the CD4+ T cell response is shown. This is not surprising, as it is due to intact virion protein antigens and may be treated as a foreign antigen and is preferably presented to CD4+ T cells by MHC class II molecules. The double fusion virus IE1/2-UL51 induces a T cell response in both the CD4+ and CD8+ phenotypes, similar to those typically observed in healthy individuals with HCMV infection.

Comparison of different formulations of double fusion virus IE1/2-UL51 and aluminum salt in Ganges The ability to produce neutralizing antibodies in monkeys (Figure 10). 30 μg/dose of double fusion virus IE1/2-UL51 was formulated with HNS (alkaline buffer), amorphous aluminum hydroxyphosphate sulfate (AAHS) or Merck aluminum phosphate adjuvant (MAPA) and at week 0 and 8 Weekly vote. Serum samples collected at week 12 showed that although MAPA enhanced neutralization antibody induction, the increase was not statistically significant (two-tailed unpaired t test).

Example 5: Identification of storage buffer

Store in HBSS (Hank's Balanced Salt Solution) with a suitable buffer and store at -70 °C until the CMV virus used is diluted approximately 10×. The residual components of the HBSS buffer in each sample included 0.533 mM potassium chloride, 0.044 mM potassium dihydrogen phosphate, 0.034 mM disodium hydrogen phosphate, 13.79 mM sodium chloride, 0.417 mM sodium bicarbonate, and 0.1% w. /v glucose. The sample is then stored at room temperature or at a temperature between 2 ° C and 8 ° C for 4 days or frozen and thawed. For freeze-thaw, the samples were stored at -70 ° C for at least 1 hour and thawed at RT for 30 minutes for one or three cycles. The stability of the test sample was tested using virus entry on day 4. Briefly, analysis was performed using several different sample dilutions to obtain a reaction curve, and EC50 (μg/mL) values were obtained from the virus entry analysis results by non-linear curve fitting. Lower EC50 values represent better stability. The EC50 values of the stability samples and the -70 °C frozen control samples were compared.

Viral entry assays measure the ability of CMV to infect ARPE-19 cells and express IE1 (ie, early protein 1). Analysis was performed in a transparent 96-well plate. Detection of target proteins in fixed cells using IE1-specific primary antibodies and biotinylated secondary antibodies, and using IR Dye 800CW Streptavidin And Sapphire 700/DRAQ5 (for cell input normalization) to quantify the fluorescent signal of each well. The results were plotted as 800/700 Integrated Intensity Ratio (Integ. Ratio) versus CMV concentration (total protein, μg/mL). EC50 values were also obtained from the results of the infectivity analysis using a non-linear curve fit. Since the viral infection of ARPE-19 cells is dependent on the integrity of the viral glycoprotein antigen, in particular the pentameric gH complex, the EC50 value reflects the extent to which the viral particles are maintained under these conditions.

As shown in Figure 11, the CMV lost its infectivity when stored in HBSS for 4 days at RT. Furthermore, when evaluated by virus entry analysis, three freeze-thaw cycles in HBSS resulted in complete loss of infectivity. Therefore, HBSS is not the optimal buffer for CMV storage.

The effect of pH on CMV room temperature stability was examined using a pH range of 3 to 8. The following buffers were used: citrate buffer (25 mM) (pH 3.0); acetate buffer (25 mM) (pH 4); acetate buffer (25 mM) (pH 5); histidine buffer (25 mM) (pH 6); HEPES buffer (25 mM) (pH 7); Hank's Balanced Salt Solution (HBSS) (pH 7.5) and Tris buffer (25 mM) (pH 8).

The sample was prepared by diluting the virus body 10 times with a suitable buffer. The samples were stored at RT (25 ° C) for 4 days. On day 4, the stability of the test sample was quantified by using virus entry analysis. The CMV stored in HBSS, which was stored frozen at -70 ° C, was treated as a control. The UV-Vis spectra of each sample were obtained at 0 and 4 days to detect structural changes and aggregation that occurred during storage.

As shown in Figure 12, 25 mM histidine buffer (pH 6) provides better stability of CMV by retaining higher infectivity at RT compared to other tested pH. Qualitative. The second derivative of the UV spectrum indicates similar structural features of the virus at all pH (data not shown). No significant aggregation was observed at any of the tested pHs, as measured by optical density at 350 nm (data not shown).

The effect of urea alone or in combination with sodium chloride on the stability of CMV virus was tested in 25 mM histidine buffer (pH 6). The addition of 2% urea alone had no effect on CMV stability. However, the combination of 2% urea and 150 mM NaCl improved the stability of CMV at RT (Figure 13).

The effect of ionic strength on CMV stability was examined at pH 6. Increasing concentrations of NaCl (0 mM, 75 mM, 150 mM, and 320 mM NaCl) were added to 25 mM histidine buffer (pH 6). CMV stability is dependent on ionic strength, with better ionic strength resulting in better stability (Figure 14). The presence of urea did not have or had the lowest effect on CMV stability (data not shown).

In addition, several other excipients (sucrose, sorbitol, glycerol, and valine) were screened for the effect of CMV stability at g at room temperature. The test vehicle to be tested was added to CMV in 25 mm histidine buffer (pH 6) at room temperature for 4 days, after which CMV virus stability was measured using virus entry assay. Calculate the EC ₅₀ value of the sample. Of all the tested excipients, 150 mM NaCl alone or in combination with 9% w/v sucrose provided better stability at pH 6 (data not shown). Therefore, the recommended CMV storage buffer has 25 mM histidine (pH 6) with 150 mM NaCl and with or without 9% w/v sucrose at RT.

The effect of cryoprotectants on CMV stability during freeze-thaw was investigated. As described above (Fig. 11), CMV stored in HBSS completely lost its infectivity when subjected to 3 freeze-thaw cycles. Screening for some low temperature protection Agents (including sucrose, sorbitol, glycerol) reduce the ability to freeze-thaw stress on CMV. For each freeze-thaw cycle, the samples were frozen at -70 °C for at least 1 hour and thawed at RT for 30 minutes. Adding a cryoprotectant increases the stability of the virus. In addition, the combination of 9% w/v sucrose and 150 mM sodium chloride significantly enhanced the stability of the virus when compared to other tested cryoprotectants (Figure 15). Therefore, the recommended CMV is stored at -70 ° C or up to 3 freeze-thaw cycles with a buffer composition of 25 mM histidine, 150 mM NaCl and 9% sucrose (HNS buffer).

HNS buffer and HBSS buffer were compared for protection against CMV stability during 3 freeze-thaw cycles, refrigeration (2-8 °C) and RT (25 °C). HNS buffer provided better stability of CMV live virus under all test storage conditions (data not shown).

Example 6: Stability of CMV in HNS Buffer

The double-fused IE1/2-UL51 CMV virus stock was supplied in HNS buffer and stored at -70 °C until use. Stability studies were performed at a concentration of 100 μg/mL based on total protein content as measured by Bradford analysis. The bulk virus was diluted with HNS buffer to obtain the final virus concentration. The samples were then stored at the appropriate temperature and tested as described for up to 3 months. For freeze-thaw, the samples were frozen at -70 °C for at least 1 hour and thawed at room temperature for 30 minutes. Samples were pulled at different time points and kept frozen at -70 °C until analysis.

The total protein content of the test samples was quantified using Bradford analysis. The total protein content of the samples did not change over the 3 month period (data not shown).

Monitor the test by measuring the hydrodynamic diameter of the sample using the DLS method The particle size of the CMV in the sample over time. This method monitors any accumulation or disruption of viral particles over time and at different storage temperatures. No real trend was observed, with some samples having sporadic changes in particle size (data not shown). The results indicate that the virions are intact and do not aggregate at elevated temperatures.

Example 7: Effect of storage conditions on viral entry and immunogenicity

Significant changes in virus entry titers (EC50 values) were observed by subjecting the CMV samples to different storage temperatures (data not shown). Storage at -20 °C resulted in lower virus entry titers compared to 2-8 °C and 25 °C. It was found that the virus at a potency of 2-8 ° C sample entered the titer below 25 ° C for storage. Based on the EC50 values, the storage temperature was graded (from most stable to most unstable) in the following order: 25 °C > 2-8 °C > -20 °C, up to 1 month time point. For samples stored at -20 ° C, 2-8 ° C, and 25 ° C, the virus entry titer was not detectable at the 3 month time point.

Mouse immunogenicity studies were initiated at the end of the stability study to determine the effect of storage temperature on the ability of CMV to induce CMV neutralizing antibodies. Mice were immunized intramuscularly with a 2.5 μg/dose vaccine on day 0 and intensified on day 21, after which blood was drawn on day 28. The CMV neutralizing antibodies against gH in mouse serum were tested using ARPE 19 cells and NT50 titers were obtained by non-linear curve fitting.

The effect of storage at different temperatures for 3 months on the immunogenicity of IE1/2-UL51 double fusion CMV was assessed. The NT50 titer was dependent on the storage temperature, with higher temperatures lowering the potency compared to the -70 °C frozen control, but not significant (p = 0.2584, one-way ANOVA) (Figure 16A). The NT50 titers of the formulations stored at -20 °C were lower, less than 1/2, but the virus entry analysis titers of these samples were significantly affected compared to the -70 °C frozen control. -20 ° C, 2-8 ° C and 25 ° C The trend of NT50 titers for stability samples follows the CMV quality ELISA titers obtained for these samples.

The effect of storage at different temperatures for 8 hours after thawing on the immunogenicity of IE1/2-UL51 double fusion CMV was assessed. The NT50 titers of the formulations were compared to the -70 °C frozen control. Storage of the samples at any of the test temperatures for 8 hours did not affect NT50 titers (p = 0.5865, one-way ANOVA) (Figure 16B).

The effect of double-fusion of IE1/2-UL51 CMV at 25 ° C for different time points after thawing the samples was evaluated in a mouse immunogenicity study. The NT50 titers of the formulations were compared to the -70 °C frozen control. Storage of the sample at 25 ° C for up to one week did not affect the NT50 titer (p = 0.1848, unpaired two-tailed t test). At 3 months, the NT 50 titer decreased to slightly below 1/2, indicating that the formulation may have stability problems at 25 ° C for a longer period of time (data not shown).

The effect of three freeze-thaw cycles on the double-fused IE1/2-UL51 CMV formulated in HNS buffer was evaluated by mouse immunogenicity. The 3 freeze-thaw (F/T) cycles of the double-fusion CMV formulation did not affect immunogenicity compared to the -70 °C frozen control (p=0.2103, unpaired two-tailed t test) (data not shown).

Other embodiments are within the scope of the following patent application. While a number of embodiments have been shown and described, various modifications may be made without departing from the spirit and scope of the invention.

1A-1B show schematic diagrams showing the construction of a CMV strain that restores the performance of the pentameric gH complex. (A) Producing a self-removing bacterial artificial chromosome (BAC) to manipulate AD 169 viral genome strategy. (B) Repair the frameshift mutation in UL131 to restore its performance. (C) Replacing GFP with the cre recombinase gene to establish a self-cleavable CMV BAC.

Figures 2A-2D show the effect of the conventional inactivation method on the immunogenicity of the gH complex. Γ-irradiation (A, B) and β-propiolactone E (BPL) (C, D) were used to deactivate CMV expressing the gH complex. The inactivation kinetics were determined by plaque assay (A, C), and the immunogenicity was determined by assessing the entry of neutralizing activity against viral epithelial cells by serum from mice administered with CMV (B, D). ).

Figure 3 shows Shield 1 concentration-dependent progeny virus production of CMVs of the gH complexes with various essential proteins fused to FKBP derivatives. ARPE-19 cells were infected with rdCMV virus for 1 h at 0.01 PFU/cell multiplicity, washed twice with fresh medium, and grown with 0 μM, 0.05 μM, 0.1 μM, 0.5 μM or 2 μM Shield-1 Cultivate in the medium. Seven days after infection, cell-free virus was collected and viral titer was determined by performing TCID50 analysis on ARPE-19 cells in the presence of 2 μM Shield 1.

Figures 4A-4D show the growth kinetics of rdCMV in ARPE-19 cells. The cells were infected with a virus containing (A) IE1/2, (B) UL51, (C) IE1/2-UL51 fusion protein or (D) parental beMAD virus at a dose of 0.01 PFU/cell. After 1 hour, the cells were washed twice with fresh medium and incubated in the absence of Shield-1 (open circles) or 2 μM Shield-1 (filled circles). Cell-free virus was collected at the indicated time points after infection, and TCID50 was performed on ARPE-19 cells in medium containing 2 μM Shield-1. Analyze to quantify infectious viruses.

Figures 5A-5E are growth kinetics of IE1/2-UL51 rdCMV in different cell types. (A) MRC-5, (B) HUVEC, (C) AoSMC, (D) SKMC, (E) CCF-STTG1 cells were infected with rdCMV virus and incubated for 1 hour. The cells were washed twice with fresh medium and subsequently incubated in the absence of Shield-1 (open circles) or 2 μM Shield-1 (filled circles). Acellular virus was collected at the indicated time points after infection, and the infectious virus was quantified by performing TCID50 analysis on ARPE-19 cells in medium containing 2 μM Shield-1.

Figures 6A-6C are immunogenicity analyses of IE1/2-UL51 rdCMV in mice, rabbits, and rhesus monkeys. (A) Mice were immunized with beMAD (open circles) or IE1/2-UL51 rdCMV (closed circles) at weeks 0 and 4. (B) Rabbits were immunized with 10 μg of beMAD or the indicated rdCMV at week 0, week 3 and week 8. (C) Immunization of rhesus monkeys with 100 μg beMAD or IE1/2-UL51rdCMV at week 0 and week 8. In each case, serum samples were collected and analyzed by performing CMV microneutralization analysis on ARPE-19 cells. The lines indicate the geometric mean neutralization titer (NT50) in each group.

Figure 7 shows the vertical axis neutralization titer of rhesus monkeys vaccinated with the double fusion virus IE1/2-UL51. At the 0th, 8th, and 24th week, the indicated doses or formulations of the rhesus monkey group (n=5) (shown as red triangles) were inoculated, while one group was at week 0, week 4, and Gb/mf59 (30 mg/dose) was received at 24 weeks. Immune sera were collected at the indicated time points and evaluated in virus neutralization assays. The NT50 of the NT50 titer was plotted on the vertical axis with the standard error of the set. AAHS: Amorphous aluminum hydroxyphosphate sulfate; IMX: ISCOMATRIX; HNS: alkaline buffer.

Figures 8A-8D show IFN-γ ELISPOT in rhesus monkeys vaccinated with 100 μg (A) or 10 μg (B-D) per dose of double fusion virus IE1/2-UL51. Do not use adjuvant (A-B), or use AAHS (C) or ISCOMATRIX (D). PBMCs were stimulated with a pool of peptides representing HCMV antigens. Gray bars represent the GMT of each antigen of this group (n=5). The responder ratio of each antigen is shown on the top of each antigen in each figure.

Figures 9A-9B show that vaccination of the double fusion virus IE1/2-UL51 in rhesus monkeys induces a T cell response in both the CD8+ (A) and CD4+ (B) phenotypes. PBMC were harvested from monkeys given a vaccine of ISCOMATRIX® as an adjuvant at a dose of 100 μg or 10 μg. PBMCs were stimulated with a pool of peptides representing HCMV antigens, followed by staining for IFN-[gamma] and CD4+/CD8+ surface T cell markers. Data are presented as the number of CD4+/CD8+ positive, IFN-[gamma] positive cells per million PBMC. Lines represent the geometric mean (GMT) of the group receiving the same vaccine (n=5). The numbers at the bottom of each figure represent the GMT of two vaccinated groups (n=10). CMV: purified virus; SEB: cell division promoter used as a positive control; IMX: ISCOMATRIX.

Figure 10 shows that Merck Aluminium Phosphate Adjuvant (MAPA) increases neutralizing antibody titers in monkeys. Rhesus monkeys were immunized with a dual fusion virus vaccine formulated in HNS (alkaline buffer), AAHS or MAPA at 30 and 10 weeks at weeks 0 and 8. Serum samples were taken at week 12 and neutralized titers were assessed. Lines represent the geometric mean of the group.

Figures 11A-11B show the stability of CMV exhibiting gH at different temperatures in Hank's Balanced Salt Solution (HBSS). (A) CMV samples stored in HBSS were stored for 4 days at the indicated temperatures, after which CMV virus stability was measured using virus entry assay. (B) Calculate the EC50 value of the sample using the virus entry analysis results. ^* Indicates that EC50 cannot be calculated due to complete loss of infectivity.

Figures 12A-12B show the effect of pH on the stability of CMV exhibiting gH at room temperature. (A) CMV samples stored in buffers with different pH were stored for 4 days at room temperature, after which CMV virus stability was measured using virus entry assay. (B) Calculate the EC50 value of the sample using the virus entry analysis results.

Figures 13A-13B show the effect of urea alone or in combination with sodium chloride on the stability of CMV virus expressing gH. (A) Add 2% urea alone or in combination with 150 mM NaCl to CMV in 25 mM histidine buffer (pH 6) and hold at room temperature for 4 days before using virus entry assay CMV virus stability. (B) Calculate the EC50 value of the sample using the virus entry analysis results.

Figures 14A-14B show the effect of ionic strength on the stability of CMV virus expressing gH. (A) Increasing concentrations of NaCl (0 mM, 75 mM, 150 mM, and 320 mM NaCl) were added to CMV in 25 mM histidine buffer (pH 6) and kept at room temperature for 4 days. Then, the virus was used to enter the assay to measure the stability of the CMV virus. (B) Calculate the EC50 value of the sample using the virus entry analysis results.

Figure 15 shows the effect of the cryoprotectant on the stability of the CMV exhibiting gH relative to the freeze-thaw cycle. The cryoprotectant shown was added to CMV in 25 mM histidine buffer (pH 6) and subjected to 3 freeze-thaw cycles, after which CMV virus stability was measured using virus entry assay. The EC50 value of the sample was calculated using the virus entry analysis results.

Figures 16A-16B show the effect of storage temperature on induction of CMV neutralizing antibodies in a mouse immunogenicity study. Mice were immunized on day 0 and intensified on day 21, after which blood was drawn on day 28. CMV neutralizing antibodies against gH in mouse serum were tested using ARPE-19 cells. NT50 titers were obtained by nonlinear curve fitting. (A) CMV samples were stored at different temperatures for 3 months prior to immunogenicity studies. (B) CMV samples were stored at different temperatures for 8 hours after thawing and prior to immunogenicity studies.

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Claims (35)

A conditionally replicating defective cell giant virus (CMV) comprising: (a) a pentameric gH complex comprising UL128, UL130, UL131, gH and gL; and (b) encoding an essential protein IE1/2 and destabilizing a first fusion protein between proteins and a nucleic acid of a second fusion protein between the essential protein UL51 and the destabilizing protein, wherein the destabilizing protein is a FK506-binding protein (FKBP) or a FKBP derivative comprising an amino acid substituted F36V And L106P, wherein wild type IE1/2 and UL51 are no longer present, and wherein the CMV is an attenuated strain which has a restored gH complex expression due to repair of a mutation in the UL131 gene. The conditional replication defective CMV of claim 1, wherein the FKBP derivative comprises an amino acid substituted FKBP of F36V and L106P. The conditional replication defective CMV of claim 1 or 2, wherein (a) the first fusion protein is SEQ ID NO: 1; and (b) the second fusion protein is SEQ ID NO: 3. The conditional replication defective CMV of claim 3, wherein (a) the first fusion protein is encoded by SEQ ID NO: 2; and (b) the second fusion protein is encoded by SEQ ID NO: 4. The conditional replication defective CMV of claim 1 wherein the genome of the CMV is SEQ ID NO: 14. The conditional replication-deficient CMV of any one of claims 1, 2, and 5, wherein the CMV is AD 169 having a restored gH complex manifested by repairing a mutation in the UL131 gene. A composition comprising the conditional replication-defective CMV of any one of claims 1 to 6 and a pharmaceutically acceptable carrier. The composition of claim 7, which further comprises an adjuvant. The composition of claim 8 wherein the adjuvant is ISCOMATRIX®. Use of a composition according to any one of claims 7 to 9 for the manufacture of a medicament for inducing an immune response against CMV in a patient. The use of claim 10, wherein the immune response is a protective immune response against CMV infection in a patient. The use of claim 10 or 11, wherein the CMV infection is a primary infection, a recurrent infection or a super-infection. The use of claim 10 or 11, wherein the patient is a human. The use of claim 13 wherein the patient's immunity is reduced. The use of claim 14, wherein the immunocompromised patient has HIV or AIDS or a transplant recipient. The use of claim 13, wherein the patient is a woman of childbearing age. Use of a conditional replication-deficient CMV according to any one of claims 1 to 6 for the manufacture of a medicament for inducing a protective immune response against CMV infection in a patient. The conditional replication-deficient CMV of any one of claims 1, 2, and 5 for use in a medicament for treating a CMV infection in a patient. A method of producing a conditional replication-deficient CMV according to any one of claims 1 to 6, which comprises propagating recombinant CMV in epithelial cells in the presence of Shield-1. The human pigmentation network of the epithelial cell line Membrane epithelial cells. The method of claim 20, wherein the human retinal pigment epithelial cell line is deposited with ARPE-19 cells of the American Type Culture Collection (ATCC) under accession number CRL-2302. The method of claim 19, wherein the Shield-1 is present at a concentration of at least 0.5 μM. The method of claim 22, wherein the Shield-1 is present in a concentration of at least 2 μM. A method of preparing a composition according to any one of claims 7 to 9 comprising: (a) breeding in an epithelial cell in a Shield-1 added medium, as in the conditional expression of any one of claims 1 to 6. Copying defective CMV; (b) collecting the conditional replication defective CMV from the culture medium; (c) substantially removing the Shield-1 from the conditional replication defective CMV; and (d) pharmaceutically acceptable A carrier is added to the conditional replication-defective CMV. The method of claim 24, wherein the epithelial cells are human pigmentary retinal epithelial cells. The method of claim 25, wherein the human retinal pigment epithelial cell line is deposited with ARPE-19 cells of the American Type Culture Collection (ATCC) under accession number CRL-2302. The method of claim 24, wherein during the step (a), the Shield-1 is A concentration of 0.5 μM was present in the medium. The method of claim 27, wherein the Shield-1 is present in the medium at a concentration of at least 2 μM during step (a). The method of claim 24, wherein the removing is by ultracentrifugation or diafiltration. The method of claim 24, wherein the removing reduces the Shield-1 to less than 0.1 μM. A composition comprising the conditional replication-deficient CMV of any one of claims 1 to 6 in a buffer having a pH between 5 and 7, the buffer comprising: (a) between 15 mM and 35 mM Between the histidine; and (b) between 100 mM and 200 mM NaCl. The composition of claim 31, further comprising between 5% and 15% sucrose. The composition of any one of clauses 31 to 32, wherein the buffer comprises 25 mM histidine and 150 mM NaCl, pH 6. The composition of claim 33, which further comprises 9% w/v sucrose. A nucleic acid of SEQ ID NO: 14.